Project Summary Eukaryotic cells have a highly conserved enzymatic system for ligating ubiquitin (Ub) to proteins. Frequently, the modification leads to degradation of the targeted protein, usually by the proteasome. Ub-protein conjugates are highly dynamic, being rapidly reversed by proteases called deubiquitylating enzymes (DUBs). Ub has many crucial roles, including important contributions to human biology. Substrates include naturally short-lived regulators and aberrant ?protein quality control? (PQC) substrates. Many human disorders, including neurodegenerative diseases, diabetes, and different cancers, are associated with abnormalities in Ub- dependent proteolysis. The Ub-proteasome system presents many drug targets for disease treatment. In this renewal, the PI proposes to extend studies on Ub-protein modification and degradation in several regulatory systems. One focus is on endoplasmic reticulum (ER)-associated degradation (ERAD) and nuclear envelope-associated degradation (NEAD). The proposed research will concentrate on the yeast Saccharomyces cerevisiae because of its experimental advantages and the strong conservation of the Ub system. Work from the PI previously identified an ERAD/NEAD Ub ligase called Doa10. There are still many outstanding questions regarding its mechanism and function. A new area of regulatory proteolysis initiated by the PI is degradation of Sts1, an essential protein that regulates both PQC at the ribosome and proteasome trafficking between the cytoplasm and nucleus. Degradation of Sts1 is intimately linked to its functional state. A final, also new research area addresses Ub system enzymes from obligate intracellular bacteria that infect humans and other species. One focus is on a DUB from Orientia tsutsugamushi, the causative agent of scrub typhus, a highly lethal disease. Initial analysis revealed the DUB has many unusual properties, including very tight binding to both Ub and clathrin adaptor proteins. The overarching goal of the proposal is to uncover the key biochemical, structural and cell biological features of these regulatory systems. The following Aims are proposed: (1) Investigate mechanistic features of the ER/NE- localized Doa10 Ub ligase, including substrate recognition and the role of Doa10 as a possible membrane-protein extraction factor, and conduct structural studies of the ligase; (2) Determine how the short-lived S. cerevisiae Sts1 karyopherin ?-binding protein is degraded and how it regulates nuclear transport, proteasome dynamics, and compartment- specific proteolysis; and (3) Examine how a Ub system enzyme expressed by the O. tsutsugamushi intracellular pathogen interferes with membrane trafficking.